Compositions containing 5α-dihydro-19-norethisterone and derivatives thereof for in vivo inhibition of aromatase

ABSTRACT

A composition for in vivo inhibition of aromatase in a mammal, which comprises an in vivo inhibitory amount of a compound having the following general formula: ##STR1## wherein R 1  is hydrogen or C 1-4  acyl, in combination with as pharmaceutically acceptable carrier or diluent thereof.

The investigations leading to this invention were supported in part byNIH research grant No. HD04945.

This is a continuation of application Ser. No. 07/019,338, filed Feb.26, 1987, now U.S. Pat. No. 4,829,059.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to compositions containing5α-dihydro-19-norethisterone (5α-DHNET) and its acyl derivatives as invivo inhibitors of the enzyme aromatase. As a result of this enzymeinhibitory effect, the present compositions can be used to prevent andtreat endocrine dependent conditions such as gynocomastia, andestrogen-dependent breast or uterine cancers.

2. Discussion of the Background

Aromatase is an enzyme which catalyzes the conversion of androgen intoestrogen, the terminal aromatization step of estrogen biosynthesis. Itis a membrane-bound complex system, which catalyzes a series of reactionsteps with multiple mono-oxygenations. Because of the complexity of thesystem, the precise pathway or reaction mechanism of aromatization hasnot yet been established, nor has the isolation of pure enzyme beenachieved. At least two distinct forms of human placental aromatase areknown. See Osawa and Higashiyama, in Microsomes, Drug Oxidations, andChemical Carcinogenesis (Coon, M. J. et al.), volume 1, pp. 225-228,Academic Press, London and New York (1980).

The product of aromatase action, estrogen, is not only essential forreproduction and development but also promotes the growth of estrogendependent cancers.

Approximately one third of the cases of human breast cancers require thefemale hormone estrogen for their growth and regress when the tumors aredeprived of the hormone. Removal of the source of estrogen is aneffective method of treating breast cancers, and otherendocrine-dependent cancers including uterine cancer.

The ovary is the major source of estrogen in premenopausal women andoophorectomy (excision of the ovary) is the classical treatment ofpremenopausal patients with advanced breast cancer. In postmenopausalpatients, the sites of estrogen biosynthesis are peripheral tissues suchas fat, skin, muscle, and the tumor itself, where the conversion ofandrogen to estrogen is catalyzed by aromatase. These peripheral tissuesare not subject to excision by surgical methods, but their aromataseactivity may be chemically inhibited by use of aromatase inhibitors.Several agents, such as aminoglutethimide, testololactone, and4-hydroxyandrostenedione, which have been used to successfully treatbreast cancer, are aromatase inhibitors. However, these drugs are notparticularly specific or highly potent, and they have deleterious sideeffects. There is, therefore, a need to develop better agents with fewerside effects for long term treatment to prevent the onset and growth ofendocrine-dependent tumors. In this respect, 5α-DHNET was found to beparticularly valuable by in vivo pharmacological studies.

Another condition associated with an imbalance in the amount of estrogenis known as gynecomastia. This is a pathological condition resulting inenlargement of the male breast. In gynecomastia, due either to normal orabnormal causes the enlargement is believed to result from disturbanceof the normal ratio of active androgen to estrogen in plasma or withinthe breast itself. In men given diethylstilbestrol, histological changesin the male breast resemble those in other forms of clinicalgynecomastia, a finding in keeping with the concept that gynecomastia isthe result of an estrogen effect. Estradiol formation in the normal manoccurs principally by the conversion of circulating androgens toestrogens in peripheral tissues. Feminization results when there is asignificant decrease in the ratio of production of testosterone toestradiol, and this may be a result of diminished testosteroneproduction or action, enhanced estrogen formation, or both processesoccurring simultaneously. Inhibitors of aromatase would have aninhibitory effect on gynecomastia when the condition is associated withenhanced estrogen formation in the male.

5α-DHNET is a reduction product of the contraceptive progestogen,norethisterone. The structure of this compound is as follows: ##STR2##wherein R, is hydrogen.

It is a known compound which has been synthesized as an intermediatefrom norethisterone by reduction with lithium in liquid ammonia (A.Bowers, H. J. Ringold and E. Denot, J. Am. Chem. Soc. 80, 6115-6118(1958)). Bowers et al reported no utility whatsoever for this compound.Unlike norethisterone, 5α-DHNET is not aromatizable by aromatase due tothe saturation of the 4-double bond and thus it does not possess theadverse consequence of being converted to an estrogenic substance afterbeing given to the patient. This compound is the major reducedmetabolite in humans of norethisterone which has been widelyadministered on a long term basis to women of reproductive age for thepast thirty years as the major ingredient of the contraceptive pill.Thus, it has been shown that the compound is safe for treatment of humansubjects even in the long term.

A number of substrate analogs have been evaluated as competitiveinhibitors of aromatase in vitro (see Schwarzel et al., Endocrinology(Baltimore) 92, 866-880 (1973)). 4-Hydroxyandrostenedione has been foundto cause regression of estrogen-dependent breast cancers of rats (Brodyet al., Endrocrinology (Baltimore) 100, 1684-1695 (1977)).

Generally, irreversible inhibitors are expected to be more effective foruse in vivo if they are targeted selectively toward the enzyme.Mechanism-based or suicide inactivators are designed to achieve a highdegree of selectivity of irreversible inhibition through a covalent-bondformation at the active site of the enzyme, but before such compoundsare acted upon by the target enzyme they are relatively unreactive andare therefore not likely to form covalent bonds with other cellularcomponents indiscriminantly. In fact, they carry a latent reactivefunctional group which is transformed through normal catalytic activityof the target enzyme into the reactive species. This activation occursafter formation of the enzyme-inhibitor complex and therefore theactivated inhibitor has a better chance to make a covalent bondconnection to a reactive group of the enzyme at or near the active siteresulting in selective inactivation of the enzyme. The compoundnorethisterone (17α-ethynyl-19-nortestosterone) is a suicide inactivatorof aromatase. 5α-DHNET has now been discovered to be an in vivoinhibitor of aromatase and it is suspected that such inhibition occursthrough a mechanism based reaction.

5α-DHNET was previously found to be a very weak in vitro aromataseinhibitor (Y. Osawa, Y. Osawa, C. Yarborough & L. Borzynski, BiochemSoc. Transactions, 656-659 (1983)), requiring a 50 μM concentration fora partial inhibition of human placental aromatase in vitro. Thiscompares to 0.0082 μM and 0.056 μM for 6α- and 6β-bromoandrostenedioneby the in vitro assay with human placental aromatase (S. J. Santner, H.Rosen, Y. Osawa & R. J. Santen, J. Steroid Biochem. 20, 1239-1242(1984)) and indicates that 5α-DHNET is a thousand-fold less potentaromatase inhibitor in in vitro assays.

The potent in vivo aromatase inhibitory activity of 5α-DHNET andsuppression of the growth and incidence of breast carcinomas inexperimental animals as shown in the following Examples were totallyunexpected and could not be deduced by inference from the previous invitro studies. Indeed, the in vitro studies showed 5α-DHNET to beessentially inactive; certainly not a candidate for in vivo activity.Thus, the properties of 5α-DHNET, which make it useful as an inhibitorof aromatase in vivo and as a drug for treatment and suppression of theincidence of certain endocrine-dependent cancers, were found for thefirst time by this invention. In general, although a variety ofcompounds have been shown to be in vitro aromatase inhibitors and havetherefore been considered potential candidates for in vivo activity, itcan now be seen that in vitro activity provides no assurance of in vivoactivity.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide compositions for thein vivo inhibition of aromatase.

Another object of this invention is to provide a method for in vivoinhibition of aromatase.

It is yet another object of the present invention to provide a method oftreatment or prevention of endocrine-related conditions.

It is yet another object of this invention to provide compositions fortreatment or prevention of endocrine-related conditions.

These and other objects as will hereinafter become more readilyapparent, have been achieved by providing compositions containing5α-DHNET and acylated derivatives thereof. Such compounds, as discussedabove, have been found to possess in vivo activity as inhibitors ofaromatase. Moreover, such compositions have been demonstrated to exertin vivo activity in appropriate animal models against endocrine-relatedcancers. In particular, breast cancer and uterine cancer are candidatesfor treatment with such compositions. Other endocrine dependentconditions such as gynocomastia, precocious puberty, endometriosis, andfeminizing adrenal tumors are similarly treatable or preventable withthe compositions of this invention. Also provided herein is a method oftreating such conditions which involves treatment of a mammal such as ahuman with an appropriate dosage amount of 5α-DHNET or a derivativethereof.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the invention and many of the attendantadvantages thereof will be readily obtained as the invention becomesbetter understood by reference to a detailed description when consideredin connection with the accompanying drawings, wherein:

FIG. 1 shows the effect of 5α-DHNET on the rat estrus cycle.

FIG. 2 shows the effect of 6β-bromoandrostenedione on the rat estruscycle.

FIG. 3 show the in vivo effect of 5α-DHNET on rat ovarian aromataseactivity.

FIG. 4 shows the in vivo effect of 6β-bromoandrostenedione on ratovarian aromatase activity.

FIG. 5 shows the in vivo effect of 6α-bromoandrostenedione on ratovarian aromatase activity.

FIG. 6 shows the effect of 5α-DHNET on the growth of NMU-induced breastcarcinomas of BUF/N rats in vivo.

FIG. 7 shows the effect of 5α-DHNET in vivo on NMU-induced breastcarcinomas of BUF/N rats.

FIG. 8 shows the administration schedule of 5α-DHNET to NMU-inducedBUF/N rats.

FIG. 9 shows the effect of 5α-DHNET and oophorectomy on breast tumorincidences of NMU-induced BUF/N rats.

FIG. 10 shows the effect of 5α-DHNET and oophorectomy on the survivalrate of BUF/N rats with NMU-induced breast carcinomas.

FIG. 11 shows the administration schedule of 5α-DHNET to NMU-inducedBUF/N rats--protocol II.

FIG. 12 shows the effect of 5α-DHNET on breast tumor incidence ofNMU-induced BUF/N rats--protocol II.

FIG. 13 shows the administration schedule of 5α-DHNET to NMU-inducedBUF/N rats--protocol III.

FIG. 14 shows the effect of 5α-DHNET on breast tumor incidence ofNMU-induced BUF/N rats--protocol III.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is based on the discovery that 5α-DHNET and itsacylated derivatives are capable of inhibiting in vivo the enzymearomatase.

The structure of 5α-DHNET and its related derivatives is as follows:##STR3## wherein R₁ =H or C₁₋₄. Acyl derivatives of 5α-DHNET may be, forexample, acetate, propionate, butyrate, etc. The acyl derivatives mayact as prodrugs; that is, the acyl moiety may be cleaved off of themolecule in vivo by the action of enzymes or otherwise, to result inactive 5α-DHNET. Thus, these acyl derivatives acting as prodrugs wouldgive rise to sustained activity. In addition, the acyl derivatives maythemselves be active against endocrine-dependent conditions and mayinhibit aromatase in vivo.

The preparation of the compounds according to the present invention maybe based on the method described in Ringold, J. Am. Chem. Soc. 80,6115-6118 (1958). Each of the reactions involved in the synthetic schememay be readily optimized by one of ordinary skill in the art based onbasic synthetic organic chemistry. The choice of solvents, temperatures,and other reaction conditions may be readily ascertained without undueexperimentation. Acylated derivatives of 5α-DHNET may be prepared bymethods of acylation which are well known to one of ordinary skill inthe area of synthetic organic chemistry.

The compounds of the present invention are notably non-toxic in vivo.For example, the minimum lethal dose of 5α-DHNET for SD rat (SPF) isgreater than 5,000 mg/kg. Accordingly, host toxicity does not appear tobe a problem with the compounds and compositions described herein.

As used herein, aromatase, or estrogen synthetase, is the enzyme complexwhich catalyzes the final step in the biosynthetic sequence fromcholesterol to the estrogens. See Bellino and Osawa, Biochemistry, 13,1925-1931 (1974). At least two forms of the enzyme complex have beenisolated from human placenta. See T. Higashiyama and Y. Osawa, Fed.Proc. Fed. Am. Soc. Exp. Biol. 42, 1836 (Abst. 458) (1983); Y. Osawa andT. Higashiyama in Microscomes, Drug Oxidations and ChemicalCarcinogenesis (M. J. Coon etal., eds.) Vol. 1, 225-228, Academic Press,London and New York (1980); Y. Osawa et al., J. Steroid Biochem. 15,4490452 (1981); Y. Osawa et al. in Microsomes, Drug Oxidations and DrugToxicity (R. Sato and R. Kato, eds.) 315-316, Japan Scientific SocietyPress, Tokyo (1982); and Y. Osawa et al., Cancer Res. 42 (Suppl),3299s-3396s (1982).

Methods for measuring the ability of 5α-DHNET and its related acylatedderivatives to inhibit aromatase in vivo are described in the exampleshereinbelow.

Mammals such as humans suffering from endocrine-related conditions,particularly estrogen-dependent breast cancers and uterine cancers, andgynocomastia, can be treated by adminstering to the patient apharmaceutically effective amount of one or more of the presentcompounds in the presence of a pharmaceutically acceptable carrier ofdiluent. Mammals suspected of being at an elevated risk of becomingafflicted by an endocrine-dependent condition, such as being a member ofa family in which other members have been afflicted by anendocrine-dependent condition, may also be treated by administering apreventive amount of a composition according to this invention.

Macklin clearly indicated in 1959 that breast cancer patients had ahigher incidence of breast cancer among their maternal and paternalgrandmothers and aunts as well as among their mothers and sisters(Macklin, M. T., Comparison of the Number of Breast Cancer DeathsObserved in Relatives of Breast Cancer Patients, and the Number Expectedon the Basis of Mortality Rates, J. Natl. Cancer Inst. 22, 927-951(1959).

According to the American Cancer Society's estimate, there will be130,000 new breast cancer patients in 1987 in the United States alone,and the chance of becoming afflicted with breast cancer for Americanwomen in their lifetime is approaching one in every ten women. Deathsdue to breast cancer are estimated to be 41,300 during 1987 in theUnited States.

Black et al in recent studies involving more than one thousand patientswith invasive breast cancers during 1970-1984 (M. M. Black and R. E.Zachrau, Family History and Hormones in Stepwise Mammary Carcinogenesis,Annals New York Academy of Sciences, 464, 367-377 (1986)), reported thatamong unselected breast cancer patients, 20-39 years of age, 47% had apositive family history (FH) of breast cancer. More specifically, 36% ofthe patients had a grandmother and/or aunt (G/A) with breast cancer. Theproportion of G/A-positive patients decreased progressively in eachsuccessive decade, down to a value of 4% among patients 60 years of ageor older. Another feature, distinctive for young G/A-positive women, isthe association with oral contraceptive usage. Sixty five percent ofG/A-positive breast cancer patients were oral contraceptive-positive incontrast to 41% of G/A-negative patients (p<0.01). The oralcontraceptives contain orally potent estrogens. It appears thatFH-associated differences exist in regard to hormone effects on themammary parenchyma of young women. The data suggest that there aresubpopulations of women whose mammary parenchyma is particularlysusceptible to malignant transformation when they are young. Suchsusceptibility is preferentially found among women whose grandmothers oraunts had breast cancer. Such familial association appears to involve anunusual sensitivity to female sex hormones in the form of oralcontraceptives. It further appears that such sensitivity is geneticallyrather than socially determined, since the involved relatives areequally likely to be paternal as well as maternal. Thus, it is suggestedthat women of reproductively active age who have FH of breast canceramong their maternal and paternal grandmothers and aunts as well asamong their mothers and sisters may be a preferable select group ofsubjects for the 5α-DHNET treatment to lower incidence of breastcancers.

A retrospective case-control study was carried out also by Black et alto determine to what degree atypical changes in the mammary duct systemare associated with increased risk of developing breast cancer (Black,Barclay, Cutler, Hankey, and Asire, Association of AtypicalCharacteristics of Benign Breast Lesions with Subsequent Risk of BreastCancer, Cancer, 29, 338-343 (1972)). Their primary finding was that awoman with some degree of ductular atypia in a benign lesion is subjectto a risk of developing breast cancer 5 times that of a woman with noevidence of atypical changes. Mammary carcinogenesis appears to be astepwise phenomenon which involves recognizable precursor lesions, i.e.,precancerous mastopathy and in situ carcinoma. Thus, women found to havesome degree of ductular atypia in a benign lesion may also be selectedas a preferable group for the 5α-DHNET treatment to prevent theprecursor lesions from transforming to invasive breast cancer.

In animal studies, while oophorectomized rats showed a heavier bodyweight compared to that of the untreated controls over a several monthperiod, the 5α-DHNET treated rats did not show any significantdifference of body weight from the control rats.

A single injection of 5α-DHNET (50 mg/kg body weight) to normal 4-daycycling rats caused a cessation of the estrus cycle for 4 to 6 cycles(16 to 24 days), whereas the sesame oil-only controls and a grouptreated with the same dose of 6β-bromoandrostenedione, a potent in vitroaromatase inhibitor (R. M. Budnick & T. L. Dao, Steroids, 35, 533-541(1980); S. J. Santner, H. Rosen, Y. Osawa and R. J. Santen, J. SteroidBiochem. 20, 1239-1242 (1984); Y. Osawa, M. J. Coon and Y. Osawa, Fed.Proc., 45, 1749, A-1564 (1986)) showed no effect on the cycle, as shownin Example 1. The four-day treatment of 4-day cycling rats with 5α-DHNETshowed, as exhibited in Example 2, a significant 67% suppression(p<0.0005) of the ovarian aromatase activity. The in vivo action of5α-DHNET was compared to those of 6α-bromoandrostenedione (onlyinsignificant 10% suppression) and 6β-bromoandrostenedione (72%suppression), both of which are potent in vitro aromatase inhibitors asshown in the references cited above.

The compounds according to the present invention are generally includedin a pharmaceutically acceptable carrier or diluent in an amountsufficient to exert a therapeutically useful in vivo inhibitory effecton aromatase.

There may also be included as part of the compositions pharmaceuticallycompatible binding agents, and/or adjuvant materials. The activematerials can also be mixed with other active materials which do notimpair the desired action and/or supplement the desired action. Theactive materials according to the present invention can be administeredby any route, for example, orally, parenterally, intravenously,intradermally, subcutaneously, or topically, in liquid or solid form.

A preferred mode of administration of the compounds of this invention isoral. Oral compositions will generally include an inert diluent or anedible carrier. They may be enclosed in gelatin capsules or compressedinto tablets. For the purpose of oral therapeutic administration, theaforesaid compounds may be incorporated with excipients and used in theform of tablets, troches, capsules, elixirs, suspensions, syrups,wafers, chewing gums and the like. The amount of active compound may bevaried depending upon the particular form.

The tablets, pills, capsules, troches and the like may contain thefollowing ingredients: a binder such as microcrystalline cellulose, gumtragacanth or gelatin; an excipient such as starch or lactose, adisintegrating agent such as alginic acid, Primogel, corn starch and thelike; a lubricant such as magnesium stearate or Sterotes; a glidant suchas colloidal silicon dioxide; and a sweetening agent such as sucrose orsaccharin or a flavoring agent such as peppermint, methyl salicylate, ororange flavoring may be added. When the dosage unit form is a capsule,it may contain, in addition to material of the above type, a liquidcarrier such as a fatty oil. Other dosage unit forms may contain othervarious materials which modify the physical form of the dosage unit, forexample, as coatings. Thus, tablets or pills may be coated with sugar,shellac, or other enteric coating agents. A syrup may contain, inaddition to the active compounds, sucrose as a sweetening agent andcertain preservatives, dyes, colorings and flavors. Materials used inpreparing these various compositions should be pharmaceutically pure andnon-toxic in the amounts used.

For the purposes of parenteral therapeutic administration, the activeingredient may be incorporated into a solution or suspension.

The solutions or suspensions may also include the following components:a sterile diluent such as water for injection, saline solution, fixedoils, polyethylene glycols, glycerine, propylene glycol or othersynthetic solvents; antibacterial agents such as benzyl alcohol ormethyl parabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. The parenteral preparationcan be enclosed in ampoules, disposable syringes or multiple dose vialsmade of glass or plastic.

While dosage values will vary with the specific severity of the diseasecondition to be alleviated or the degree of risk of the patient ofcontracting an endocrine-dependent condition, good results are achievedwhen the compounds described herein are administered to a subjectrequiring such administration as an effective oral, parenteral orintravenous dose of from 1 to 500 mg/day per patient. A particularlypreferred effective amount is about 5 to 250 mg/day per patient. A mostpreferred effective amount is about 10 to 150 mg/day per patient. Thesedosage ranges are what is meant by an in vivo aromatase inhibitoryamount or an anti-(endocrine-dependent condition) amount of the presentcompositions. It is to be understood, however, that for any particularsubject, specific dosage regimens should be adjusted to the individualneed and the professional judgment of the person administering orsupervising the administration of the aforesaid compound. It is to befurther understood that the dosages set forth herein are exemplary onlyand they do not limit the scope or practice of the invention. Thedosages may be administered at once, or may be divided into a number ofsmaller doses to be administered at varying intervals of time.

The invention now being generally described, the same will be betterunderstood by reference to certain specific examples which are includedherein for purposes of illustration only and are not intended to belimiting of the invention or any embodiment thereof, unless specified.

EXAMPLES Overview of Examples

The effect of 5α-DHNET on the growth of endocrine-dependent breastcancers was assessed in BUF/N rats with breast carcinomas induced byN-nitrosomethylurea (NMU). 5α-DHNET at 50 mg/kg body weight/day wasgiven daily for three weeks to rats already having at least one tumor, 2cm or larger. The total volume and number of tumors were measured. Theresults showed, as provided in Example 3, that 5α-DHNET significantlysuppressed the growth of breast carcinomas while untreated animalsshowed a rapid tumor growth. The preventive effect of 5α-DHNET on theincidence and development of breast cancers was assessed by givingcarcinogen primed animals 5α-DHNET for a one week period at 50 mg/kgbody weight/day before any breast tumor was detectable. Oophorectomy isknown to lower the incidence and death rate due to breast carcinomas inthis animal model (P. M. Gullino, H. M. Pettigrew, and F. H. Grantham,J. Natl. Cancer Institute, 54, 401-409 (1975)). As shown in Examples4-6, 5α-DHNET given at different times was found in all cases tosignificantly lower the incidence of breast carcinoma and the death rateas effectively as complete oophorectomy.

An observation at the 98th day after the initial NMU injection under thefirst protocol of 5α-DHNET treatment for the prevention of breastcarcinoma development, as described in Example 4, gave a tumor incidenceof 13% (2/16) for the 5α-DHNET treated group compared to 100% (16/16)for the control group and 38% (6/16) for the oophorectomized group. Thedeath rate observed at the 180th day after the first injection of NMUwas 25% (4/16), 31% (5/16), and 69% (11/16) for the 5α-DHNET treated,oophorectomized, and control groups, respectively. These in vivostudies, therefore, indicate that 5α-DHNET may be effectively and safelyused for the prevention and treatment of breast cancers.

Biological Evaluation of 5α-DHNET EXAMPLE 1 Effect of 5α-DHNET on theEstrus Cycle

BUF/N inbred female rats (50-80 days old) showing regular 4-day estruscycles were selected by a daily vaginal smear test. A fine powder of thesteroid sample was suspended in sesame oil (30 mg/ml) and injectedsubcutaneously on the proestrus day at a single dose of 50 mg/kg bodyweight. Vaginal smears were examined daily until normal estrus returnedfor at least three cycles. The results from the single injection of5α-DHNET are given in FIG. 1 and those for 6β-bromoandrostenedione aregiven in FIG. 2 for comparison. 5α-DHNET caused a cessation of theestrus cycles for at least four cycles on all of the animals tested(4/4). In contast, 6β-bromoandrostenedione, a known potent in vitroaromatase inhibitor, failed to affect the estrus cycle.

EXAMPLE 2 In Vivo Effect of 5α-DHNET on the Ovarian Aromatase Activity

5α-DHNET (30 mg/ml sesame oil suspension) was injected s.c. twice dailyfor four days at a dose of 50 mg/kg body weight/day to BUF/N inbredfemale rats (50-80 days old) showing regular 4-day estrus cyclesstarting on the estrus day. The control group received injections ofsesame oil in the same manner. Both ovaries were removed 3-4 hours afterthe final injection. The 105,000 xg precipitate fraction of the ovarianhomogenate was used for the aromatase assay. Aromatase activity wasdetermined by the ³ H-water method (F. L. Bellino, S. S. H. Gilani, S.S. Eng, Y. Osawa and W. L. Duax, Biochemistry, 15, 4730-4736 (1976)).The sedimented ovarian enzyme preparation was homogenized in 0.1Mphosphate buffer (pH 7.5) and incubated with [1β-³ H, 4-¹⁴C]androstenedione (3.18×10⁵ dpm ³ H, 1.18×10⁴ dpm ¹⁴ C, 100.2 ng), NADPH(1 mg) and BSA (5 mg) in 0.1M phosphate buffer (pH 7.5) in a totalvolume of 1.5 ml for 30 min at 37° C. Trichloroacetic acid solution (0.2ml of 10% aqueous solution) was added to stop the enzyme reaction.Activated charcoal was added to the mixture and the spun supernatant waspassed through a cotton plugged disposable pipet. The filtrate wascounted for simultaneous ³ H and ¹⁴ C in a liquid scintillationspectrophotometer. The ³ H-water released from the 1β-³ H labeledandrogen quantitatively corresponds to estrogen formation. The resultsof the in vivo aromatase suppression assay with 5α -DHNET,6β-bromoandrostenedione (6β-BRA), and 6α-bromoandrostenedione (6α-BRA)are given in FIGS. 3, 4, and 5, respectively. 5α-DHNET suppressed 67% ofthe ovarian aromatase, whereas 6β-bromoandrostenedione suppressed 72%and 6α-bromoandrostenedione only an insignificant 10%.

EXAMPLE 3 Effect of 5α-DHNET on Growth of Breast Carcinomas

The animal model for human mammary carcinomas developed by Gullino etal. (P. M. Gullino, H. M. Pettigrew, and F. H. Grantham, J. Natl. CancerInst., 54, 401-414 (1975)) was used for this evaluation of 5α-DHNET.N-Nitrosomethylurea (NMU), wet with 3% acetic acid, was dissolved indistilled water (10 mg/ml) and given in three intravenous injections ata dose of 50 mg/kg body weight to BUF/N inbred female rats of 50 days ofage on the first injection. The second injection was 4 weeks and thethird injection was 8 weeks after the initial administration. The onsetof tumors was monitored by daily visual inspection and by palpation ofthe mammary regions three times a week. The increase in tumor size wasmonitored by vernier caliper measurements of the subcutaneous mass.One-half the average of the longest and shortest tumor diameters wastaken as the value of r in the formula 4/3 πr³ used to estimate tumorvolume. 5α-DHNET in sesame oil was given i.p. daily at a dose of 50mg/kg body weight/day for 3 weeks to BUF/N female rats having at leastone tumor, 2 cm or larger. The number and size of the tumors weremonitored on 12 treated and 13 vehicle-only control animals. The meaninitial volume of the tumors was 5.10 and 5.53 ml for the control andtreated group, respectively. The results are given in FIGS. 6 and 7 andin Table 1. While the tumors in the control group grew very rapidly,growth arrest was clearly observed in 75% (9/12) of the treated group.The death rate was lower in the treated group (3/12 in 4 weeks) than inthe control (5/13 in 3 weeks). After stopping the administration of5α-DHNET, approximately half of the responded animals were maintainedwithout a growth of tumors for another 3 weeks while the remaindershowed a slow rate of tumor growth. Averaged relative tumor volume iscompared in FIG. 7. This also indicates the suppression of tumor growthby the 5α-DHNET treatment. A summary of observations on each tumor atthe end of treatment is given in Table 1. While 24 new tumors developedin 9 control rats in 3 weeks, only 7 developed in 11 treated rats. While98% of tumors in the control rats grew and only 2% (1/53) regressedduring the 3 week period, the treated group showed that 13% of theinitially observed tumors had disappeared in the 3 week period and 42%of the tumors had regressed.

                                      TABLE 1                                     __________________________________________________________________________    EFFECT OF 5α-DHNET ON THE NUMBER OF NMU-INDUCED                         TUMORS IN BUF/N RATS                                                                     Total No. of Tumors                                                       No.of     newly                                                               Rats                                                                              start                                                                            3 wk                                                                             developed                                                                           growing                                                                            disappeared                                                                         regressed                                   __________________________________________________________________________    Control                                                                               9  29 53 24    52   0      1                                                                 52/53       1/53                                                              (98%)      (2%)                                        5α-DHNET                                                                       11  47 48  7    28   6     20                                                                 28/48                                                                              6/47  20/48                                                              (58%)                                                                              (13%) (42%)                                       __________________________________________________________________________

EXAMPLE 4 Effect of 5α-DHNET on the Incidence of Breast Carcinoma

The preventive effect of 5α-DHNET to lower the incidence of breastcarcinoma was assessed by use of the same animal model as in Example 3but by modification of the administration protocol. The first protocolof 5α-DHNET administration and oophorectomy (OVX) is given in FIG. 8.The vehicle-only control was prepared as described in Example 3.Oophorectomy was carried out at the 4th week. 5α-DHNET at a dose of 50mg/kg body weight/day was given i.p. daily for one week starting at the4th week. The onset of tumors, their number and location in each animalwere recorded daily. The results are given in FIGS. 9 and 10 and inTable 2. The control group developed tumors in 100% (16/16) of theanimals by the 98th day after the first NMU injection. At this pointtumor incidence for the 5α-DHNET treated and oophorectomized groups was13% (2/16) and 38% (6/16), respectively. Both groups gradually increasedthe incidence and by the 180th day after the first NMU injection the5α-DHNET and OVX group developed breast carcinomas in 50% (8/16) and 56%(9/16) respectively, as shown in FIG. 9. The death rate of the 5α-DHNETand oophorectomized groups were also significantly lower than that ofthe control. At the 180th day the death rate for the 5α-DHNET treatedand oophorectomized group was 25% and 31%, respectively, compared to 69%for the control group. The mean latent period of tumor development was72, 101, and 116 days for the control, OVX, and 5α-DHNET group,respectively. The average number of tumors per tumor carrying animal was6.6, 2.0, and 1.8 for the control, OVX, and 5α-DHNET group, also showingthat when 5α-DHNET was administered a long time prior to the detectionof the tumor, it significantly supressed the development of the breastcarcinomas.

                  TABLE 2                                                         ______________________________________                                        PREVENTIVE EFFECT of 5α-DHNET ON NMU-INDUCED                            TUMORS IN BUF/N RATS                                                                                             Average                                                                       No. of                                                                Mean    tumors per                                                            Latent  tumor-                                            % Tumor             Period  carrying                                          Incidence                                                                              Death      (days)  rat                                        ______________________________________                                        Control  16/16 (100%)                                                                             11/16 (69%)                                                                               72   6.6                                      OVX      9/16 (56%) 5/16 (31%) 101   2.0                                      5α-DHNET                                                                         8/16 (50%) 4/16 (25%) 116   1.8                                      ______________________________________                                    

EXAMPLE 5 Effect of 5α-DHNET on the Incidence of Breast Carcinoma

The preventive and suppressive effect of 5α-DHNET on breast carcinomaswas further examined in order to ascertain that this agent is effectiveunder general conditions rather than limited to a specificallycontrolled protocol. Only a single dose of NMU was given to the BUF/Nrats at 50th day of age and an oophorectomy was carried out at the 6thweek after the NMU injection. The 5α-DHNET group received the agent forone week starting at the 6th week at a dose of 50 mg/kg body weight/dayas given in FIG. 11. Monitoring was carried out as described in Example4. The results are given in FIG. 12. After 41 weeks of observation, thetumor incidence was 65%, 33%, and 38% for the control, 5α-DHNET, and OVXgroup, respectively. The average number of tumors per tumor carrying ratwas 2.6, 1.5, and 1.0 for the control, 5α-DHNET, and OVX group,respectively.

EXAMPLE 6 Effect of 5α-DHNET on the Incidence of Breast Carcinoma

The protocol was further modified to evaluate the generality of theeffect of 5α-DHNET on breast carcinomas. As shown in FIG. 13, the5α-DHNET treatment was given to rats 3 weeks after a single dose NMUinjection. Oophorectomy was also carried out at 3 weeks after the NMUinjection. The results are given in FIG. 14. After 37 weeks ofmonitoring, the tumor incidence was 87%, 38%, and 19% for the control,5α-DHNET, and OVX group, respectively. The average number of tumors pertumor carrying animal was 2.8, 1.5 and 1.0 for the control, 5α-DHNET,and OVX group, respectively. The results indicate that 5α-DHNET given atany stage is effective in suppressing the development of breastcondition and can be used in a general manner.

Obviously, numerous modifications and variations of the presentinvention are possible in light of the above teachings. It is thereforeto be understood that within the scope of the appended claims, theinvention may be practiced otherwise than as specifically describedherein.

What is claimed as new and desired to be secured by letters patent ofthe United States is:
 1. A composition for in vivo inhibition ofaromatase in a mammal, which comprises an in vivo aromatase inhibitoryamount of a compound having the following general formula: ##STR4##wherein R₁ is C₁₋₄ acyl, in combination with a pharmaceuticallyacceptable carrier or diluent thereof.
 2. The composition of claim 1,wherein said composition is formulated for oral use.
 3. The compositionof claim 1, wherein said compound is included in said composition suchthat a unit dosage amount of said composition is 1 mg to 500 mg per dayper patient.
 4. A method of treatment or prevention of anendocrine-dependent condition in a mammal, which comprises administeringto a mammal afflicted with an endocrine-dependent condition or in dangerof becoming afflicted with an endocrine-dependent condition, ananti-(endocrine-dependent condition) effective amount of a compositioncontaining a compound of the following general formula: ##STR5## whereinR₁ is C₁₋₄ acyl, in combination with a pharmaceutically effectivecarrier or diluent.
 5. The method of claim 4, wherein said mammal is ahuman.
 6. The method of claim 4, wherein a unit dosage form of saidcomposition delivers from 1 to 500 mg per day per patient of saidcompound.
 7. The method of claim 4, wherein said composition isformulated for oral administration.
 8. The method of claim 4, whereinsaid endocrine-dependent condition is breast cancer.
 9. The method ofclaim 4, wherein said endocrine-dependent condition is uterine cancer.10. The method of claim 4, wherein said endocrine-dependent condition isselected from the group consisting of gynocomastia, precocious puberty,endometriosis and feminizing adrenal tumor.
 11. A method of inhibitingaromatase in vivo, which comprises administering to a mammal in need ofsaid inhibition, a composition containing an active ingredient havingthe following general formula: ##STR6## wherein R₁ is C₁₋₄ acyl, incombination with a pharmaceutically acceptable carrier or diluent. 12.The method of claim 11, wherein said mammal is a human.
 13. The methodof claim 11, wherein said composition is administered orally to saidmammal.
 14. The method of claim 11, wherein a unit dosage form of saidcomposition containing from 1 to 500 mg per day per patient.